Method for determining DNA nucleotide sequence

ABSTRACT

A method for sequencing a target DNA fragment in which along with amplification of the target DNA fragment, nucleic acid transcripts are generated using an RNA polymerase and the amplified target DNA fragments are used as templates in the presence of terminators for nucleic acid transcription reaction and the generated nucleic acid transcripts are analyzed, characterized in that the amplification of target DNA fragments and the generation of nucleic acid transcripts are carried out at a constant temperature is disclosed. The amplification of target DNA fragments and the generation of nucleic acid transcripts can be carried out around the room temperature. A DNA sequencing method using a novel method in which without using a thermo-resistant RNA polymerase, the amplification of target DNA fragments and generation of nucleic acid transcript can be carried out simultaneously in parallel is provided.

RELATED FIELDS

The present invention relates to a method for DNA sequencing utilizingthe strand displacement amplification method. The present inventionrelates to a method of DNA sequencing using RNA polymerase in whichamplification of a target DNA fragment and preparation of ribonucleotidefragment for DNA sequencing can be performed in parallel withoutvariation of the temperature.

BACKGROUND TECHNOLOGY

Polymerase chain reaction (PCR) is an excellent method, and its field ofapplication increases every year (Randall K. Saiki et al. (1988) Science239, 487-491). In PCR, it is also possible to amplify DNA fragmentstarting with just of 1 molecule. A method in which an amplified productof PCR is sequenced without cloning (direct sequencing method) is alsouseful (Corinne Wong et al. (1988) Nature, 330, 384-386). This methodrequires neither preparation nor screening of a library, and it is arapid method enabling to simultaneously obtain sequence information ofmultiple samples.

Moreover, the inventor introduced a completely novel DNA sequencingmethod which does not require to remove remaining unreacted primers and2′deoxyribonucleoside5′triphosphate (2′ dNTPs), and which does notrequire denaturation so that the problem of quick regeneration of PCRproducts could be obviated [WO96/14434]. This method is a directsequencing method using RNA polymerase such as T7 RNA polymerase andterminators for RNA transcription reaction (e.g.3′deoxyribonucleoside5′triphosphate, 3′dNTPs).

The above-mentioned direct transcription sequencing method is performedas described below. RNA polymerase is reacted in a mixture ofribonucleoside5′triphosphates (NTPs) and deoxyribonucleotide(s) (3′dNTP(s)) using DNA amplified by PCR method and the like as a template.In this reaction, ribonucleotides having the bases corresponding to thetemplate DNA sequence are incorporated into a ribonucleotide sequence,termination occurs within corporation of 3′deoxyribonucleotide, and as aresult a polynucleotide is synthesized. Resulted polyribonucleotides(nucleic acid transcription products) are separated, and the DNAsequence is determined by analyzing nucleic acid sequence of theseparated fraction. Specifically, using florescence labeled 3′ dNTPderivatives as a terminator of nucleic acid transcription, nucleic acidsequence can be easily determined by analyzing the label which has beenincorporated as a part of the terminator.

By this method, the nucleic acid sequence of PCR-amplified DNA productscan be directly used for sequencing without having to remove primers and2′deoxyribonucleoside5′triphosphates (2′ dNTPs). This is because 2′dNTPs do not work as substrates for RNA polymerase. Furthermore, sinceno denaturation is required, the problem of quick regeneration of thePCR products can be avoided. Therefore the method is extremely powerful.

In the case that a large amount of nucleotide sequence such as the humangenome is to be analyzed, a method much more rapid and easier than theexisting methods is necessary in order to obtain results in a shorttime. The above-mentioned direct transcript sequencing method is arelatively rapid method compared to previous sequencing methodsutilizing DNA polymerase, however an even more rapid and easier methodis necessary. Therefore, it can be thought that a DNA amplification withpolymerase chain reaction and a nucleic acid transcript reaction maytake place in parallel in the same reaction solution using the abovedirect transcript sequencing method enabling sequencing rapidly andeasily.

However, thepolymerase chain reaction requires an increase or decreaseof the temperature of the reaction solution for an amplification of DNAfragments. Therefore, use of thermo-resistant DNA polymerase is requiredfor the polymerase chain reaction. Thus RNA polymerase used for nucleicacid transcript reaction is also required to be thermo-resistant. Theabove combination method will be possibly performed if athermo-resistant RNA polymerase having the thermo-resistance similar tothat of DNA polymerase would be available. However, at present suchthermo-resistant RNA polymerase is not known.

Therefore, an object of the present invention is to provide a method forsequencing DNA in which target DNA amplification and nucleic transcriptgeneration can be operated simultaneously in parallel without use ofthermo-resistant RNA polymerase.

SUMMARY OF THE INVENTION

The present invention relates to a method for sequencing a target DNAfragment in which along with amplification of the target DNA fragment,nucleic acid transcripts are generated using an RNA polymerase and theamplified target DNA fragments are used as templates in the presence ofterminators for nucleic acid transcription reaction and the generatednucleic acid transcripts are analyzed, characterized in that theamplification of target DNA fragments and the generation of nucleic acidtranscripts are carried out at a constant temperature (the firstmethod).

The present invention also relates to a method for sequencing DNAcomprising

a step of obtaining nucleic acid transcripts while DNA fragmentscomprising the target DNA fragment sequence are being amplified

by allowing

(g) a DNA polymerase and

(h) a RNA polymerase to work in the presence of

(a-1) a DNA fragment comprising the target DNA fragment sequence whereinthe DNA fragment comprises a sequence accepting formation of a nick andon at least one strand, a promoter sequence for a RNA polymerase,

(b) a primer comprising a primer sequence for one strand of the targetDNA fragment and a sequence accepting formation of a nick (hereinafterreferred to primer G1)

(c) a primer comprising a primer sequence for the other strand of thetarget DNA fragment and a sequence accepting formation of a nick(hereinafter referred to primer G2),

provided that at least one of the primers G1 and G2 comprises thepromoter sequence for the RNA polymerase,

(d) deoxyribonucleoside-5′-triphosphates comprising dATP, dGTP, dCTP anddTTP or derivatives thereof (hereinafter referred to dNTP derivatives),

(e) ribonucleoside-5′-triphosphates comprising ATP, GTP, CTP and UTP orderivatives thereof (hereinafter referred to NTP derivatives), and

(f) 3′-deoxyribonucleoside-5′-triphosphates comprising 3′dATP, 3′dGTP,3′dCTP and 3′dUTP or derivatives thereof (hereinafter referred to 3′dNTPderivatives),

and by forming a nick at a site of the DNA fragment (a-1) acceptingformation of a nick; and

a step of separating the resulting nucleic acid transcripts and readingthe nucleic acid sequence from the separated fractions (the secondmethod).

The present invention further relates to a method for sequencing a DNAcomprising

a step in which primer B1 (a primer complementary to one strand of thetarget DNA fragment), primer B2 (a primer complementary to the otherstrand of the DNA fragment), primer G1, and primer G2 hybridize to theDNA fragment (provided that the primer B1 hybridizes to a site closer to5′ end of one strand of the DNA fragment than the site recognized byprimer G1, and the primer B2 hybridizes to a site closer to 5′ end ofthe other strand of the DNA fragment than the site recognized by primerG2), and

a step of obtaining nucleic acid transcripts while DNA fragmentscomprising the target DNA fragment sequence are being amplified, byallowing (g) a DNA polymerase and (h) a RNA polymerase to work on thetarget DNA (a-2) obtained from the hybridization in the presence of (b)primer G1, (c) primer G2, (d) dNTP derivatives, (e) NTP derivatives and(f) at least one kind of 3′ dNTP derivatives and by forming a nick at asite of the DNA fragment (a-2) accepting formation of a nick; and

a step of separating the resulting nucleic acid transcripts and readingthe nucleic acid sequence from the separated fractions (The thirdmethod).

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is an electrogram of nucleic acid obtained from the Example.

MODES FOR CARRYING OUT THE INVENTION

Methods of the present invention are briefly composed of a step in whicha DNA fragment comprising the target DNA fragment sequence is amplifiedand nucleic acid transcript products are produced using amplified DNAfragments as a template, and a step in which obtained nucleic acidtranscript products are separated and nucleic acid sequence isdetermined from the separated fraction.

In the first method, along with amplification of a target DNA fragment,nucleic acid products are produced by means of RNA polymerase using theamplified target DNA fragments as a template in the presence ofterminators of a nucleic acid transcript reaction, and sequencing theresulting nucleic acid sequence of target DNA fragments by analyzinggenerated nucleic acid transcripts. This method is characterized in thatthe amplification of said target DNA fragment and production of nucleicacid transcript are operated at a constant temperature. The “constanttemperature” herein means that the reaction temperature is not tointentionally be increased or decreased. Amplification of a target DNAfragment and production of nucleic acid transcripts can be conductedaround the room temperature, and the temperature used can be selectedconsidering an optimum temperature of enzymes in use such as RNApolymerase.

The amplification of a target DNA fragment can be carried out by using,for example, the strand displacement amplification (SDA) method. The SDAmethod is described in Walker et al., Nucleic Acids Research, 1992Vol.20, No.7, 1691-1696. The SDA method is a method in which the DNApolymerase and restriction enzymes are allowed to work on a target DNAfragment in the presence of substrates of DNA polymerase and primers forthe target DNA fragment. The above primers consist of a combination ofsense direction and antisense direction, both primers include a sequencein which a nick can be formed by restriction enzymes. Moreover, at leastone primer comprises a promoter sequence for RNA polymerase.

In addition, a method of producing nucleic acid transcripts by RNApolymerase utilizing a target DNA fragment as a template in the presenceof terminators of a nucleic acid transcript reaction and analyzing theresulting nucleic acid transcripts can be a method in which the nucleicacid transcription reaction is conducted in the presence of substratesfor RNA polymerase and labeled terminators, and the analysis of nucleicacid transcripts is carried out by detection of the labels which havebeen incorporated into the nucleic acid transcripts. Examples of suchmethods include a direct transcript sequencing method utilizing RNApolymerase such as T7 RNA polymerase and RNA transcript reactionterminators (for example, 3′-deoxyribonucleoside-5′triphosphate, 3′dNTPs) described in WO96/14434.

The second and the third method are embodiments of the first method.

Step of Amplification and Nucleic Transcription

In the second method, a DNA fragment (a-1) comprising a target DNAfragment sequence is used as template of amplification, and this DNAfragment comprises a sequence accepting nick formation and a promotersequence for RNA polymerase on at least one strand.

In the third method, a target DNA fragment (a-2) hybridized with primerB1, primer B2, primer G1, and primer G2 is used as template foramplification.

The “target DNA fragment” is a fragment to be analyzed for its nucleicacid sequence. There is no special limitation to the kind and length ofthe target DNA fragment.

The “sequence accepting nick formation” is, for example, “a restrictionenzyme site comprising a hemiphosphorothioate site or analogue thereof”.The “restriction enzyme site comprising a hemiphosphorothioate site” isthat one of the nucleotides of the restriction enzyme site being an αSbody (1-thiotriphosphate). If a restriction enzyme reacts at arestriction enzyme site comprising hemiphosphorothioate, the nucleotidecomplementary to the as body is cleaved and a nick is introduced (onlyone side of the strand is cleaved). And, if necessary a sequence ofrestriction enzyme site can be determined according to the kind ofrestriction enzyme used for nick formation described below.

“Promoter sequence for RNA polymerase” is a promoter sequence recognizedby RNA polymerase used for the nucleic acid transcription reactiondescribed below. The promoter sequence for RNA polymerase is selectedaccording to the kind of RNA polymerase used.

DNA fragment (a-1) comprising a target DNA fragment sequence, a sequenceaccepting nick formation and a promoter sequence for RNA polymerase canbe prepared, for example, by the method described below.

The above sequence is prepared by a method comprising a step ofhybridizing the primer B1, primer B2, primer G1 and primer G2 to the DNAfragment, a step where DNA polymerase is allowed to work on the targetDNA fragment in the presence of dNTP derivatives. Primer B1 is a primerwhich binds to one strand of the target DNA sequence, and primer B2 is aprimer which binds to the other strand of target DNA sequence, primer G1is a primer comprising a primer sequence for one strand of target DNAfragment and a sequence accepting nick formation, and primer G2 is aprimer comprising a primer sequence for the other strand of the targetDNA fragment and a sequence accepting nick formation. Provided that theprimer B1 hybridizes at a position closer to the 5′end of one strand ofthe target DNA fragment than the primer G1, and the primer B2 hybridizesat a position closer to the 5′end of the other strand of the target DNAfragment than the primer G2.

Hybridization of primers to the DNA fragment can be performed by, forexample, heat treatment at 95° C. for 4 minutes, followed by slowcooling. The sequences of primer B1 and primer G1 are selected such thatprimer B1 can hybridize to 5′upstream compared to primer G1. Inaddition, each primer sequence is selected such that the primers B1 andG1 hybridized to the DNA fragment are separated by a single strandregion on the hybridized products. Similarly, as for primer B2 andprimer G2, each primer sequence is selected such that primer B2hybridizes to 5′ upstream, and the primers B2 and G2 hybridized to theDNA fragment are separated by a single strand region on the hybridizedproducts. Each primer can be suitably synthesized by conventionalmethods.

These strands to which the primer B1 and primer G1 or the primer B2 andprimer G2 have hybridized can be amplified by the strand displacementamplification method using DNA polymerase in the presence of dNTPs. Thestrand displacement amplification method is described in Walker et al.,Nucleic Acids Research, 1992 vol.20, No.7, 1691-1696. Moreover, in theabove amplification, if αS body is utilized for one of dNTP derivatives,a restriction site comprising a hemiphosphorothioate site can be formed.In addition, the selection of the αS body of dNTP derivatives can besuitably determined according to a sequence of restriction site.

In the third method, the target DNA fragment (a-2) hybridized to theprimer B1, primer B2, primer G1 and primer G2 is used as a template foramplification. The “target DNA fragment” is the fragment to be analyzedfor its nucleic acid sequence. There is no special limitation to thekind and length of the target DNA fragment. The Primer B1, primer B2,primer G1 and primer G2 are as described above, and as for primer B1 andprimer G1, the sequences are selected such that primer B1 hybridizes to5′ upstream compared to primer G1. Moreover, each primer sequence isselected such that a single strand region exists between the primers B1and G1 on the hybridized products. Similarly, as for the primer B2 andprimer G2, each primer sequence is selected such that primer B2hybridizes to 5′ upstream compared to primer G2 and a single strandregion exists between primer B2 and primer G2 on the hybridizedproducts. Further, each primer can be suitably synthesized byconventional methods. Hybridization of primers to the target DNAfragment can be conducted by, for example, heat treatment at 95° C. for4 minutes, followed by gradual cooling.

In the third method, a DNA fragment (a-2) comprising the target DNAfragment sequence (provided that this DNA fragment comprising a sequenceaccepting nick formation and a promoter sequence for RNA polymerase inat least a single strand) is produced by DNA polymerase as describedabove using the hybridization products as a template in the beginning ofamplification and nucleic acid transcript step.

In the amplification and the nucleic acid transcription step, (g) DNApolymerase and (h) RNA polymerase are allowed to work on the (a-1) or(a-2) target DNA fragment in the presence of (b) primer G1, (c) primerG2, (d) dNTP derivatives (provided that, one of dNTP derivatives is αSbody), (e) NTP derivatives and (f) at least one of 3′dNTP derivatives,and a nick is formed at a site accepting nick formation on the DNAfragment (a-1) or (a-2). This reaction is conducted at a substantiallyconstant temperature without increasing or decreasing the temperature.However, the reaction temperature is suitably determined based on theoptimum temperatures of the each enzyme. Nick formation on DNA fragments(a-1) and/or (a-2) can be performed with restriction enzymes. Somerestriction enzymes form a nick by recognizing, for example, ahemiphosphorothioate site locating on a restriction enzyme site.Furthermore, a sequence accepting nick formation can be a restrictionenzyme site which comprises a hemiphosphorothioate site or an analogoussite thereof, and one of dNTP derivatives used at the time is a αS bodyor a similar compound.

In the above described reaction system, reactions catalyzed by DNApolymerase, restriction enzymes and RNA polymerase operate in parallel.

The restriction enzyme cleaves a sequence accepting nick formation, forexample, a restriction site comprising a hemiphosphorothioate sitecomprised in (a-1) or (a-2) target DNA fragment and forms a nick at therestriction site of the DNA fragment. It is generally known that when arestriction enzyme, which recognizes a restriction site comprising ahemiphosphorothioate site, works on the restriction site, a nick isformed on a strand complementary to a strand having ahemiphosphorothioate site only on one of strands. In the presentinvention, primer chains G1 and G2 do not contain hemiphosphorothioate.Thus among restriction sites composed of the primers existing on doublestrand, only one strand has a hemiphosphorothioate sites, and thereforea nick is formed on such a restriction site. However, even if the samerestriction site exists in the target DNA fragment, since sites which donot comprise one of the primer chains have hemiphosphorothioate sites onboth of strands, a nick unnecessary for the present purpose will not beformed.

Further, a nick formation activity of restriction enzymes variesdepending on the kind of the enzymes, therefore a restriction enzymeused can be selected in view of its activity. HincII, BstBI, Aval andthe like can be used as a restriction enzyme without any limitation.Moreover, suitable restriction enzymes is available from commercialenzymes.

DNA polymerase amplifies a sequence by strand displacement amplificationreaction which starts from a nick formed on a DNA fragment by therestriction enzyme and uses as a template the DNA fragment to which anick has been formed. dNTP derivatives are used as substrates.Furthermore, when a sequence accepting nick formation is a restrictionsite comprising hemiphosphorothioate, a hemiphosphorothioate site can beformed on such restriction site of the DNA fragment by amplificationusing as-body as one of dNTP derivatives. αS-bodies of dNTP derivativesare commercially available. Further, based on the sequence of therestriction site, one can select dNTP to become the αs-body.

DNA polymerase used is not specially limited, and can be suitablyselected from DNA polymerases which can readily catalyze a stranddisplacement amplification reaction. Examples of the DNA polymeraseinclude Bst pol, exe klenow and the like, but not limited to these. TheDNA polymerases are also commercially available.

RNA polymerase uses a DNA fragment amplified by DNA polymerase as atemplate, and produces a nucleic acid transcript by using (e) four kindsof NTP derivatives having different bases and (f) at least one kind of3′ dNTP derivative as substrates.

The synthesis of RNA or nucleic acid is terminated by incorporation of3′ dNTP derivatives into the 3′ end of the RNA or nucleic acidtranscript since the 3′ dNTP derivatives lack the 3′ hydroxy-residue. Asa result, RNA or nucleic acid fragment with different length having 3′dNTP derivatives at 3′ ends are produced. Suchdeoxyribonucleotide-analogues can be obtained for each of the four kindsof 3′ dNTP derivatives having different nucleotide. The four kinds ofdeoxyribonucleotide-analogues can be used to determine the RNA ornucleic acid sequence [Vladimir D. Axelred et al. (1985) BiochemistryVol. 24, 5716-5723]. 3′ dNTP derivatives preferably comprises a labelfor the purpose of a sequence analysis. Examples of the labels of 3′dNTP derivatives include florescence, radioactive or stable isotopes. 3′dNTP derivatives labeled with a stable isotope are commerciallyavailable. Further, labeled 3′ dNTP derivatives can be synthesized byusing known methods [for example, WO96/14434].

In the above mentioned nucleic acid transcription reaction, it ispreferred that the sequence with respect to all (four) kinds of nucleicacids can be determined by only one operation in which a transcriptionreaction is performed using four different 3′ dNTP derivatives havingdifferent nucleic acid each are labeled with a different label, theresulting nucleic acid transcription reaction products are separated,and the nucleic acid sequence is determined by analyzing the signalsfrom the different labels of the obtained separated fractions.

Therefore, four kinds of transcription products with different 3′ dNTPderivatives at 3′ ends can be obtained by four nucleic acidtranscription reactions using four different 3′ dNTP derivatives. Ineach of the nucleic acid transcription reactions, any one of 3′ dNTPderivatives is used. However, this method is not efficient.

In the second method, a nucleic acid transcription step can be performedby using a DNA fragment (a-1) of which one strand comprises a sequenceaccepting nick formation and a promoter sequence for RNA polymerase, oneof the primer G1 and G2 which comprises a promoter sequence for an RNApolymerase (the promoter sequence is preferably the same as the sequencewhich is comprised in the DNA fragment (a-1)), and a RNA polymerasewhich can be activated by the promoter sequence. In this case, thesequence is determined by reading the sequence of only one strand of thetarget DNA fragment.

It is possible to determine the sequences by reading both strands of thetarget DNA fragment. In this case, the used DNA fragment (a-1) comprisesa sequence accepting nick formation and a promoter sequence for RNApolymerase in both strands. Moreover, primers G1 and G2 each comprisinga promoter sequence for RNA polymerase are used. Provided that, thepromoter sequence comprised in the primer G1 is identical with one ofthe promoter sequences comprised in one strand of the double strand DNAfragment (a-1). The promoter sequence comprised in the primer G2 isidentical with the other of the promoter sequences comprised in theother strand of the double strand DNA fragment (a-1). In addition, thenucleic acid sequence analysis comprising a step of nucleic acidtranscription is performed by using one of two RNA polymerases which isactivated by only one of the two promoter sequences. Analysis data withrespect to each strand of the target DNA fragment can be obtained byconducting the nucleic acid sequence analysis comprising a step ofnucleic acid transcription using different RNA polymerases. The nucleicacid sequence of the target DNA fragment can be determined based on theresulting two kinds of analysis data. In this case, the readingaccurateness can be advantageously improved since determination of thenucleic acid sequence is conducted independently with respect to eachstrand of the target DNA fragment, and the sequence is determined basedon the resulting sequences with respect to two strands.

The RNA polymerase can either be a wild-type RNA polymerase or a mutantRNA polymerase. The mutant RNA polymerase is preferably a modifiedwild-type RNA polymerase of which at least one amino acid has beenmodified to improve its 3′ dNTP derivatives incorporation activitycompared to that of the wild type RNA polymerase. The “wild type RNApolymerase” herein includes all RNA polymerase existing in the nature,moreover, it can also be a modified wild type RNA polymerase which hassubstitution, insertion or deletion of amino acids which are not themodification for obtaining increased activity for incorporating3′-deoxylibonucleotide or its derivative in comparison with thecorresponding wild type RNA polymerase. That is, wild type RNApolymerases artificially modified with a purpose other than thatdescribed above are included in the above “wild-type RNA polymerase”. Itis suitable to make such a substitution, insertion or deletion of aminoacids to the extent that the activity of RNA polymerase is maintained.

Examples of the mutant RNA polymerase include mutant T7 RNA polymerasesF644Y and L665P/F667Y. The numbers indicate an amino acid numbercounting from the N terminal of the polymerase protein. For example,F667 means that the amino acid residue No. 667 is F, and F667Y meansthat the amino acid residue F No. 667 is substituted by Y. These sustainthe RNA synthesis activity sufficiently and have an improved ability forincorporating 3′ dNTPs. The strong bias observed in the wild-type hasbeen considerably decreased.

Mutant RNA polymerases can be prepared using general recombinant DNAtechnology. Further, E. coli strain pT7RF644Y (DH5 α) andpT7RL665P/F667Y (DH5 α), which produce the mutant T7 RNA polymeraseF644Y and L665P/F667Y respectively, were deposited at the NationalInstitute of Bioscience and Human-Technology (NIBH) with internationaldeposition numbers 5998 (FERM-BP-5998) and 5999 (FERM-BP-5999) on Jul.2^(nd), 1997.

A system in which the amplification and nucleic acid transcriptionreaction of a DNA fragment comprising the target DNA fragment sequenceare performed simultaneously in parallel is described above. However, itis possible to sequentially perform a DNA fragment amplificationcomprising the target DNA fragment sequence using the stranddisplacement amplification method first, and a nucleic acidtranscription reaction using the obtained DNA fragment as a template.However, as described above, it is simple and efficient tosimultaneously perform a DNA fragment amplification and nucleic acidtranscription reaction in one reaction vessel in parallel.

Separation and Detection of Nucleic Acid Transcript

In the method of the present invention, a nucleic acid transcriptionproduct is separated. The separation can be suitably performed by anymethod which enables the separation of numerous product molecules havingdifferent molecular weight, included in the transcription productsaccording to the molecular weight. Examples of such methods includeelectrophoresis. HPLC can also be used.

Conditions of electrophoresis and the like are not particularly limitedand it can be carried out in a conventional manner. The sequence of RNAor nucleic acid can be determined from bands (nucleic acid ladder)provided by subjecting the transcription products to electrophoresis.

RNA or nucleic acid ladders can be read by detecting labels of 3′ dNTPderivatives which have been incorporated in the transcription reactioninto each fragment. More precisely, a sequence of the transcript can bedetermined by detecting radioactive or stable isotope atom, orflorescence of obtained bands which result from an electrophoresis oflabeled transcripts. For the detection of ladders generatingradioactivity or stable isotope atom, or florescence, for example, asystem used for DNA sequencing can suitably be used.

From the RNA or nucleic acid sequence determined as above, DNA sequenceused for the template of transcript can be determined. When a ladder isformed for each nucleic acid, DNA sequence used as a template of thetranscription can be determined by integrating the information of RNA ornucleic acid sequence provided from four kinds of ladders. Further, whenladders are formed for two or more nucleic acids (bases) simultaneously(in the case that two or more nucleic acid bands are present in the sameladder), DNA sequence used as a template for the transcription can bedetermined by integrating the RNA or nucleic acid sequence informationobtained from each of the ladders. In particular, when a ladder issimultaneously formed for four kinds of nucleic acids (in the case thatfour kinds of nucleic acids bands are present in a ladder), DNA sequenceused as a template for the transcription can be determined from RNA ornucleic acid sequence information obtained from the ladder.

The method of the present invention is a DNA sequencing method utilizinga novel method which can perform a target DNA amplification and apreparation of nucleic acid transcripts at the same time in parallel. Inthe present invention, a target DNA amplification and preparation ofnucleic acid transcripts, which have been conventionally conductedindependently, can be performed simultaneously in parallel, thereforenucleic acid sequencing of DNA can be done more efficiently than theconventional method.

EXAMPLE

The present invention is further illustrated in following example.

A sequencing method of the present invention is used for a specific siteof human p53 gene used for diagnosis of mutant cancer and the like.

Material

1) Preparation of Primers

Preparation of primers: four kinds of primers were prepared near p53exon 8 referring to following references (1) and (2).

Reference (1) Relating to SDA

Walker, G. T., Fraiser, M. S., Schram, J. L., Little, M. C., Nadeau, J.G. and Malinowski, D. P. Nucleic Acids res. 20 (1992) 1691-1696. Stranddisplacement amplification—an isothermal, in vitro DNA amplificationtechnique.

Walker, G. T., Little, M. C. Nadeau, J. G. and Shank, D. D. Proc. Natl.Acad. Sci. USA 89 (1992) 392-396. Isothermal in vitro amplification ofDNA by a restriction enzyme/DNA polymerase system.

Reference (2) Relating to Nucleic Acid Sequence of Human p53

Buchman, L. L. Chumakov, P. M., Ninkina, N. N., Samarina, O. P. andGiorgiev, G. P. Gene 70 (1988) 245-252. A variation in the structure ofthe protein-coding region of the human p53 gene.

Primer B1: 5′-CCTATCCTGAGTA (13mers, nucleic acid sequence No.1408-1420) (SEQ ID NO: 1)

Primer B2: 3′-TGATTCAGAACCC (13mers, complement to nucleic acid sequenceNo. 1664-1676) (SEQ ID NO: 2)

Primer G1:5′-CGAATCGTTGTCTCGGGGCATAATACGACTCACTATAGGGCCCAATCTACTGGGAC(SEQ ID NO:3) (The first underlined site from 5′end: a restriction site; the secondunderlined site: promoter site for T7 RNA polymerase; the thirdunderlined site: 13 nucleic acids; complement to p53 nucleic acidsequence No. 1227-1338)

Primer G2: 3′-TCTTATAAAGTGG GGGCTCTTCAGACCTCGCCTTAGC (SEQ ID NO: 4) (Thefirst underlined site from 3′end: 13 nucleic acids: complement to p53nucleic acid sequence No. 1643-1655, the second underlined site:restriction site)

2) Enzyme

Restriction enzyme BsoBI: purchased from New England Biolab. This enzymecleaves C/PyCGpuG sequence. DNA polymerase (Bst po1): purchased fromEpicentre. T7 RNA polymerase (mutant, F644Y) : mutant T7 RNA polymerasein which F (phenylalanine) residue No. 667 was replaced by Y (tyrosine)residue wild type T7 RNA polymerase.

3) DNA

Human placental DNA and Ht-29 cell line from human large intestinecancer (obtained from ATCC) are used. The p53 DNA of said cancer cellline is known to contain a replacement of its amino acid R by H at No.273 of exon site 8 (G→A replacement).

See Reference (3) Murakami, Y., Hayashi, K., Hirhashi, S. and Sekiya, T.Cancer Res. 51 (1991) 5520-5525. Aberration of the tumor suppressor p53and retinoblastoma genes in human hepatocellular carcinomas.

Murakami, Y., Hayashi, K. and Sekiya, T. Cancer Res. 51 (1991)3356-3361. Detection of aberrations of the p53 alleles and the genetranscript in human tumor cell lines by single-strand conformationpolymorphorism analysis.

Method

(1) The following materials were mixed and modified at 95° C. for 4minutes, and then primer annealing was performed at 37° C. 15 for oneminute.

0.5 μg/ml human placental DNA or human cancer cell line DNA . . . 1 μl

Each primer G1 and G2 (10 μM) . . . 1 μl

Each primer B1 and B2 (1 μM) . . . 1 μl

dGTP, dATP, dTTP (2 mM each) . . . 2 μl

d αSCTP (10 mM) . . . 2 μl

10×buffer * . . . 2 μl

GTP, ATP, UTP, CTP (2 mM each) . . . 5 μl

Water . . . 1 μl

four color florescence labeled nucleotide mixture ** . . . 1 μl

(notes: * 500 mM NaCl, 100 mM Tris-HCl (pH 7.5), 100 mM MgCl₂, 10 mM DTTsolution; ** a mixed solution of R11C-3′dGTP 1 μM, R6G-3′dATP 1 μM,ROX-3′dCTP 50 μM, TMR-3′dUTP 12.5 μM; R11C-3′dGTP, R6G-3′dATP,R0X-3′dCTP and TMR-3′dUTP are florescence labeled 3′dNTP)

(2) The following materials were added to the mixture of (1) to give afinal volume of 20 μl, and allowed to react at 45° C. for 2 hours.

Restriction enzyme BsoBI (10 units/μl) . . . 1 μl

DNA polymerase (Bst po1) 5 units/μl . . . 2 μl

T7 RNA polymerase (F644Y) 25 units/μl . . . 1 μl

(3) The reaction solution is analyzed by electrophoresis for sequencing.

FIG. 1 shows an electrogram of nucleic acid sequences which correspondto a part of p53 exon of human placental DNA (upper) and human cancercell line HT29 DNA (lower). As seen from the figure, in the upperfigure, amino acid at 273 is R(CGT) , on the other hand, in the lowerfigure, half of them are the amino acid H(CAT).

In the present invention, nucleic acid sequencing can be performedthrough a DNA amplification in the same cube at the same temperatureafter only once heat denaturation.

4 1 13 DNA Artificial Sequence Description of Artificial Sequence 5′primer B1; 13mers complementary to nucleic acids 1408-1420 of human p53gene 1 cctatcctga gta 13 2 13 DNA Artificial Sequence Description ofArtificial Sequence 3′ primer B2; 13 mers complementary to nucleic acids1664-1676 of p53 human gene 2 cccaagactt agt 13 3 56 DNA ArtificialSequence promoter (21)..(40) promoter site for T7 RNA polymerase 3cgaatcgttg tctcggggca taatacgact cactataggg cccaatctac tgggac 56 4 37DNA Artificial Sequence Description of Artificial Sequence 3′ primer G24 cgattccgct ccagacttct cgggggtgaa atattct 37

What is claimed is:
 1. A method for sequencing a target DNA fragmentcomprising amplifying the target DNA fragment, generating nucleic acidtranscripts from the target DNA fragment using an RNA polymerase in thepresence of terminators for nucleic acid transcription reactions, anddetermining the sequence of said nucleic acid transcripts, wherein saidamplifying and generating are carried out at a constant temperature. 2.The method according to claim 1, wherein the amplification of target DNAfragments and the generation of nucleic acid transcripts are carried outat around room temperature.
 3. The method according to claim 1, whereinthe amplification of target DNA fragments is carried out by the stranddisplacement amplification method.
 4. The method according to claim 3,wherein the strand displacement amplification method comprises combininga DNA polymerase and a restriction enzyme with the DNA fragment in thepresence of substrates for the DNA polymerase and two primers for thetarget DNA fragment wherein the primers comprise a restriction enzymerecognition sequence.
 5. The method according to claim 4 wherein one orboth of the primers comprises a promoter sequence for the RNA polymerasein addition to the restriction enzyme recognition sequence.
 6. Themethod according to claim 1, wherein the nucleic acid transcription iscarried out in the presence of substrates for the RNA polymerase andlabeled terminators, wherein the step of determining the sequence ofsaid nucleic acid transcripts is carried out by detecting.
 7. A methodfor sequencing DNA comprising obtaining nucleic acid transcripts whileDNA fragments comprising a target DNA fragment sequence are beingamplified, by combining a DNA polymerase and a RNA polymerase with a DNAfragment (a-1) comprising the target DNA fragment sequence wherein theDNA fragment (a-1) comprises a sequence accepting formation of a nickand on at least one strand, a promoter sequence for a RNA polymerase, aprimer (G1) comprising a primer sequence for one strand of the targetDNA fragment and a restriction enzyme recognition sequence, a primer(G2) comprising a primer sequence for the other strand of the target DNAfragment and a sequence accepting formation of a nick, provided that atleast one of the primers G1 and G2 comprises the promoter sequence forthe RNA polymerase, deoxyribonucleoside-5′ triphosphates comprisingdATP, dGTP, dCTP and dTTP or derivatives thereof, ribonucleoside-5′triphosphates comprising ATP, GTP, CTP and UTP or derivatives thereof,and 3′ deoxyribonucleoside-5′ triphosphates comprising 3′ ATP, 3′ GTP,3′ CTP and 3′ UTP or derivatives thereof, forming a nick at a site ofthe DNA fragment (a-1) accepting formation of a nick; separating thenucleic transcripts; and determining the nucleic acid sequence of saidnucleic acid transcript.
 8. A method according to claim 7, wherein thetarget DNA fragment (a-1) is prepared by a method comprising hybridizinga primer (B1) for one strand of the target DNA fragment, a primer (B2)for the other strand of the target DNA fragment, a primer G1 and aprimer G2 to the DNA fragment in the presence of dNTP derivatives,wherein primer B1 hybridizes to a site closer to 5′ end of one strand ofthe DNA fragment than the site recognized by primer G1, and primer B2hybridizes to a site closer to 5′ end of the other strand of the DNAfragment than the site recognized by primer G2, and combining a DNApolymerase with the DNA fragment.
 9. A method according to claim 7,wherein one strand of the target DNA fragment (a-1) comprisesrestriction enzyme recognition sequence and a promoter sequence for RNApolymerase, one of the primers G1 and G2 comprises a promoter sequencefor the RNA polymerase, wherein this promoter sequence has the samesequence as that comprised in the DNA fragment (a-1), and the nucleicacid transcription is carried out using an RNA polymerase, wherein theactivity of the RNA polymerase is initiated by the promoter sequence.10. A method according to claim 7, wherein both strands of the DNAfragment (a-1) comprise restriction enzyme recognition sequence and apromoter sequence for an RNA polymerase, each of the primers G1 and G2comprises a promoter sequence for RNA polymerase, wherein the promotersequence comprised in the primer G1 has the same sequence as one of thepromoter sequences comprised in the DNA fragment (a-1), the promotersequence comprised in the primer G2 has the same sequence as the otherof the promoter sequences comprised in the DNA fragment wherein, whereinthe step of determining the nucleic acid sequence comprises determiningthe sequence of both strands of the DNA fragment (a-1) independentlyusing two types of RNA polymerase, wherein each RNA polymerase onlyrecognizes one of the two promoter sequences.
 11. A method of target DNAsequencing comprising hybridizing primers B1, B2, G1, and G2 to a DNAfragment (a-1) comprising a target DNA fragment, to produce a DNAfragment (a-2); obtaining nucleic acid transcripts, while simultaneouslyamplifying DNA fragments (A-2) comprising the target DNA fragment andperforming an extension reaction, by combining a DNA polymerase and aRNA polymerase a DNA fragment (a-2) the primer B1, the primer G2, dNTPderivatives, NTP derivatives and at least one kind of 3′ dNTPderivatives, and forming a nick at a site of the DNA fragment (a-2)restriction enzyme recognition sequence; separating the nucleic acidtranscripts and determining the nucleic acid sequence of said nucleicacid transcripts.
 12. A method according to claim 7, wherein the nickformed on the DNA fragment (a-1) is formed using a restriction enzyme.13. A method according to claim 7, wherein the restriction enzymerecognition sequence is a restriction site comprising ahemiphosphorothioate site or an analogous site thereof, and one of thedNTP derivatives is an αS derivative or an analogue thereof.
 14. Amethod according to claim 7, wherein the obtaining of nucleic acidtranscripts and the amplification of DNA fragment (a-2) are carried outat a substantially constant temperature.
 15. A method according to claim7, wherein the 3′ dNTP derivatives are labeled.
 16. A method accordingto claim 15, wherein the label is a fluorescent substance, or aradioactive or stable isotope element.
 17. A method according to claim7, wherein four types of 3′ NTP derivatives different in bases andbearing different labels from each other are used for the nucleic acidtranscription reaction, and the nucleic transcripts are separated fromone another and a nucleic acid sequence is determined from signalsobtained from the labels of the separated nucleic acid transcripts.